The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

The effect of maltose on dextran yield and molecular weight distribution

Dextran synthesis has been studied since the Second World War, when it was used as blood plasma expander. This polysaccharide composed of glucose units is linked by an α-1,6-glucosidic bond. Dextransucrase is a bacterial extra cellular enzyme, which promotes the dextran synthesis from sucrose. When, besides sucrose, another substrate (acceptor) is also present in the reactor, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products, decreasing the dextran yield. Although dextran enzymatic synthesis has been extensively studied, there are few published studies regarding its molecular weight distribution. In this work, the effect of maltose on yield and dextran molecular weight synthesized using dextransucrase from Leuconostoc mesenteroides B512F, was investigated. According to the obtained results, maltose is not able to control and reduce dextran molecular weight distribution and synthesis carried out with or without maltose presented the same molecular weight distribution profile.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

On the production of dextran by free and immobilized dextransucrase

Dextransucrase from Leuconostoc mesenteroides was produced in a semicontinuous culture with slow addition of a concentrated sucrose solution. The resulting high activity of the fermentation broth allowed a one-step purification method, by gel permeation chromatography (GPC) in 96.4% yield. This procedure resulted in 140-fold purification, with specific activity of 122 U/mg. The enzyme was immobilized onto an amino-Spherosil support activated with glutaraldehyde. Preparations with dextransucrase activities as high as 40.5 U/g of support were obtained, when low specific area supports were used and maltose was added during the enzyme coupling. Diffusional limitations were found during enzyme reaction, as shown by a kinetic study. As a consequence of immobilization, the average molecular weight of dextrans seems to increase. Immobilized dextransucrase looks promising for low-molecular-weight dextran production. Clinical dextran was synthesized when the polysaccharides produced in the presence of maltose were used as acceptor of a second synthesis reaction. The molecular weight distribution of the resulting production was less disperse than when clinical dextran was produced by acid hydrolysis of high-molecular-weight dextran.     

The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran-cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.     

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.     

Molecular Characteristics of Bovine Serum Albumin-Dextran Conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5–1 mol of dextran could be bound to 1 mol of native BSA. SDS–PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95–2.35×10⁵ g/mol and a less-abundant fraction with aggregates greater than 1.50×10⁶ g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.     

Structure of the linear, low molecular weight dextran synthesized by a d-glucosyltransferase (GTF-S3) of Streptococcus sobrinus

Four extracellular α-d-glucosyltransferases (GTF) have been separated from cultures of Streptococcus sobrinus strains grown in continuous culture. Three of the GTF synthesized soluble dextrans from sucrose, and one of these enzymes, GTF-S3, catalysed the production of a low molecular weight, linear dextran. Methylation analysis and high field proton NMR spectroscopy on the intact S3 glucans confirmed that these dextrans were small (dp 20–30) and linear, with the majority of chains terminated with a sucrosyl moiety. Enzymic hydrolysis, followed by analytical and semi-preparative HPLC, led to the isolation of only linear oligosaccharides, one of which was identified as 6^G-glucosylsucrose.The results are accommodated by the two-site insertion mechanism for dextran synthesis proposed by Robyt et al. (1974) (Arch. Biochem. Biophys., 165, 634).     

Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD–CD2

GBD–CD2 is an α-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the α-1,2 transglucosylation of glucosyl moieties onto α-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD–CD2 follows a Ping Pong Bi Bi mechanism with a high k _cat value of 970 s⁻¹. The amount of the synthesised α-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled α-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of α-1,2 linkages ever reported.     

Physicochemical studies of oligodextran. I. Molecular weight dependence of intrinsic viscosity, partial specific compressibility and hydrated water

The number average molecular weight, Mn, of low molecular weight dextran was determined through endgroup analysis, and the intrinsic, viscosities of these materials in aqueous solution were determined at 25°C. The ultrasonic velocities in their aqueous solutions were also measured at 25 and 45°C. As concerns the molecular weight dependence of the intrinsic viscosity, partial specific compressibility of solute and the hound water around the solute, the following results were obtained. (1) log [η]-log Mn and [η]/Mn^0.5 – Mn^0.5 plots were in accord with the Mark-Houwink and Stockmayer-Fix-man equations respectively for Mn > 2, 000, but these plots deviated from the equations for Mn < 2, 000. (2)The partial specific compressibility, β ₁°, of dextran is expressed by following equation for Mn < 2,000: β ₁° = 10^?12 × (13.6 log Mn - 51.7) (cm²/dyne). In contrast, it, becomes the constant value, -- 7.3 × 10^?12 cm²/dyne, for Mn > 2,000. (3) The amount of bound water of dextran calculated from the sound velocity measurement lakes constant value of 0.17 ml g for Mn > 2, 000, but the amount of hydration increase with decreasing molecular weight for Mn < 2,000. From these results, a dextran molecule in aqueous solution is expected to change its conformation from random coiling to uncoiling stretched form at the molecular weight of around 2, 000 or about 12 glucose units.     

Multiple Forms of Inorganic Pyrophosphatase from the Leaf of Amarantus blitum L.

The existence of a carrier-bound pathway for inorganic sulfate assimilation has been proposed in Chlorella and Escherichia coli. The possibility that the sulfonyl group of active sulfate is transferred to a specific organic acceptor to form thiosulfate ester was examined with Salmonella typhimurium LT-2. Some 11% of the radioactive products from [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate were transferred to high molecular weight compounds, and the remainder of the product is identified as free inorganic sulfite. Apparent thiosulfonate reductase activity was detected in the reaction mixtures containing S-sulfoglutathione and NADPH as conceivable substrates, but not with partially purified sulfite reductase. The former activity was attributable to the nonenzymatic reaction, sulfitolysis. Through these in vitro experiments the existence of the carrier-bound pathway was disproved.     

Gelation of Limulus Lysate by Synthetic Dextran Derivatives

The Limulus test has been considered specific for the presence of bacterial endotoxins. To synthesize a simple model of endotoxin, palmitoyldextran phosphate was prepared by modification of dextran by palmitoylation and phosphorylation. The present studies indicated that a variety of polysaccharide derivatives, such as palmitoyldextran phosphate, palmitoyldextran, and dextran phosphate, give a positive Limulus test and show pyrogenic activity, except for low molecular dextran derivatives. On the other hand, polysaccharides, such as dextran, starch (soluble), chitosan, xylan, and lentinan, were negative in these assays. The gelation reaction of Limulus lysate by modified dextran derivatives may depend on the molecular weight or modification of polysaccharides by palmitoylation and/or phosphorylation to a great extent.     

Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes

The water-insoluble 1,4-β-linked products formed from UDP-[¹⁴C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [¹⁴C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[¹⁴C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-β-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc₄Xyl₃, which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-α-d-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[¹⁴C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-β-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.     

Lipase-mediated Transformation of Vegetable Oils into Biodiesel using Propan-2-ol as Acyl Acceptor

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 °C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7^th cycle when methanol was used as an acyl acceptor, under standard reaction conditions. Revisions requested 22 December 2005; Revisions received 26 January 2006     

Tunicamycin Inhibits Biosynthesis of Acid Mucopolysaccharides in Cultures of Chick Embryo Fibroblasts

Enterobacter cloacae KY 3074 grown in a medium containing xanthine, hypoxanthine, guanine, or their nucleosides and nucleotides produced xanthine oxidase. The purified enzyme preparation showed a major protein band and a few minor bands in acrylamide gel electrophoresis. Molecular oxygen was the most effective electron acceptor. Ferricyanide and 2,6-dichlorophenolindophenol also served as electron acceptors, but NAD and NADP did not. Xanthine and hypoxanthine were good substrates, and guanine was also an effective substrate. The activity was inhibited by Ag²⁺, Cu²⁺, PCMB, and ascorbate. The spectrum of the Enterobacter enzyme resembled that of some known xanthine oxidizing enzymes, and this suggests a similarity in the prosthetic groups of these enzymes. The molecular weight of the native enzyme and subunit was 128,000 and 69,000, respectively.     

Glycogen synthesis by cell-free extracts from the dimorphic yeast Candida albicans

A particulate glucosyltransferase prepared from budding and filamentous cultures of Candida albicans used uridine diphosphate glucose as sole glucosyl donor in a reaction (measured by following the incorporation of [¹⁴C]-glucose from UDP [¹⁴C]-glucose into polymer) stimulated by glucose-6-phosphate and inhibited by adenosine triphosphate and guanosine triphosphate. The radiolabelled reaction product was solubilized by -amylase, and, on oxidation with periodate followed by reduction with borohydride and acid hydrolysis, yielded erythritol and glycerol in the ratio of 4 to 1. The radiolabelled glucosyl residues were attached to an endogenous acceptor of high molecular weight.     

A novel dextransucrase is produced by <Emphasis Type="Italic">Leuconostoc</Emphasis><Emphasis Type="Italic">citreum</Emphasis> strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives

The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240 kDa and a soluble dextransucrase (68.46%) at 160–180 kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and ¹³C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.     

Purification,properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of >1,092 molxmin^-1xmg protein^-1. The enzyme is a hexamer of a polypeptide of Mr=42,500, and the native molecular weight is 250,800. The apparent K _m values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD⁺ in the deamination reaction, and 7.2 mM for -ketoglutarate, 243 mM for NH ₄ ⁺ and 0.028 mM for NADH in the reverse reaction. NADP⁺ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate -ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.     

Rapid Purification and Crystallization of Thermostable Malate Dehydrogenase Isolated from a Thermophilic Bacterium,Thermus flavus AT 62

A structural study of the water-soluble dextran made by Leuconostoc mesenteroides strain C (NRRL B-1298) was conducted by enzymic degradation and subsequent ¹³C-NMR analysis of the native dextran and its limit dextrins. The α-l,2-debranching enzyme removed almost all of the branched D-glucose residues, and gave a limit dextrin having a much longer sequence of the internal chain length (degree of linearity: n = 24.5 compared with the value of n = 3.3 for the native dextran). The degree of hydrolysis with debranching enzyme corresponded to the content of α-1,2-linkages determined by chemical methods, which suggested that most of the α-l,2-linkages in the dextran B-1298 constituted branch points of a single D-glucose residue. A synergistic increase of susceptibility of the dextran B-1299 was observed by simultaneous use of debranching enzyme and endodex-tranase. ¹³C-NMR spectral analysis indicated the similarity of structure of dextran B-1298 to that of B-1396, rather than that of B-1299. Occurrence of α-l,3-linkages in the limit dextrin was supported by a newly visualized chemical shift at 83.7 ppm.